ASTM E1153-22

Standard Test Method for Efficacy of Sanitizers Recommended for Inanimate, Hard, Nonporous Non-Food Contact Surfaces

SIGNIFICANCE AND USE
4.1 This test method shall be used to determine if a chemical intended for use as a non-food contact sanitizer or as a one-step cleaner-sanitizer provides percent reductions of the selected test organisms on treated carriers as compared to control.
SCOPE
1.1 This test method is used to evaluate the antimicrobial efficacy of sanitizers on precleaned, inanimate, hard, nonporous, non-food contact surfaces against Staphylococcus aureus, or Klebsiella pneumoniae or Klebsiella aerogenes, or a combination thereof. Appropriate modifications to the method may be required when testing organisms not specified herein. When utilizing test surfaces not described herein (see Test Method E2274) or when evaluating spray-based or towelette-based antimicrobial products, modifications may also be required.  
1.2 This test method may also be used to evaluate the antimicrobial efficacy of one-step cleaner-sanitizer formulations recommended for use on lightly soiled, inanimate, nonporous, non-food contact surfaces.  
1.3 It is the responsibility of the investigator to determine whether Good Laboratory Practices (GLP) are required and to follow them where appropriate (see section 40 CFR, 160 or as revised.)  
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 This standard may involve hazardous materials, chemicals and microorganisms and should be performed only by persons who have had formal microbiological training.  This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Published
• Publication Date: 30-Sep-2022
• ICS: 11.080.20 - Disinfectants and antiseptics
• ICS: 71.100.35 - Chemicals for industrial and domestic disinfection purposes
• Technical Committee: E35 - Pesticides, Antimicrobials, and Alternative Control Agents
• Drafting Committee: E35.15 - Antimicrobial Agents

Relations

• Refers: ASTM E2756-19 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-Nov-2019
• Refers: ASTM E2756-18 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-Apr-2018
• Refers: ASTM E2274-16 - Standard Test Method for Evaluation of Laundry Sanitizers and Disinfectants
• Effective Date: 15-Apr-2016
• Refers: ASTM E2756-10 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-May-2010
• Refers: ASTM E2274-09 - Standard Test Method for Evaluation of Laundry Sanitizers and Disinfectants
• Effective Date: 01-Nov-2009
• Refers: ASTM E1054-08 - Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents
• Effective Date: 01-Apr-2008
• Refers: ASTM D1193-06 - Standard Specification for Reagent Water
• Effective Date: 01-Mar-2006
• Refers: ASTM E2274-03 - Standard Test Method for Evaluation of Laundry Sanitizers and Disinfectants
• Effective Date: 01-Dec-2003
• Refers: ASTM E1054-02 - Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents
• Effective Date: 10-May-2002
• Refers: ASTM D1193-99e1 - Standard Specification for Reagent Water
• Effective Date: 10-Feb-1999
• Refers: ASTM D1193-99 - Standard Specification for Reagent Water
• Effective Date: 10-Feb-1999

Overview

ASTM E1153-22 is the latest revision of the "Standard Test Method for Efficacy of Sanitizers Recommended for Inanimate, Hard, Nonporous Non-Food Contact Surfaces." Developed by ASTM International, this standard provides a method for evaluating the antimicrobial effectiveness of chemical sanitizers and one-step cleaner-sanitizer formulations. It specifically applies to products intended for use on precleaned, hard, nonporous surfaces that are not intended for contact with food. The primary microorganisms tested under this method are Staphylococcus aureus, Klebsiella pneumoniae, and Klebsiella aerogenes (formerly Enterobacter aerogenes).

Key Topics

• Antimicrobial Efficacy Assessment: The standard outlines methodologies to measure the percent reduction of selected bacteria on treated surfaces compared to controls, ensuring products deliver reliable performance as sanitizers.
• Test Organisms: Targeted organisms include Staphylococcus aureus, Klebsiella pneumoniae, and Klebsiella aerogenes, which are relevant pathogens for evaluating surface sanitizing agents.
• Surface Types: Surfaces tested must be inanimate, hard, nonporous, and non-food contact, such as glass, stainless steel, or glazed ceramic.
• One-Step Cleaner-Sanitizers: The method can be adapted to assess the efficacy of formulations designed to both clean and sanitize lightly soiled surfaces in a single step.
• Lab Practices and Safety: Emphasizes the importance of Good Laboratory Practices (GLP), formal microbiological training, and compliance with health and safety guidelines when handling chemicals and microorganisms.
• Reporting and Reproducibility: Specifies the type of data to be reported, including percent reductions, product details, and testing parameters, but notes that precision and bias must be evaluated by individual laboratories due to inherent variability.

Applications

ASTM E1153-22 is widely used by:

• Manufacturers of Sanitizers: To substantiate antimicrobial claims for products intended for non-food contact applications in institutional, industrial, commercial, and residential settings.
• Regulatory Bodies: As a test reference for product registration and compliance where antimicrobial efficacy needs to be demonstrated.
• Quality Control Laboratories: To ensure ongoing effectiveness of sanitizing agents and cleaner-sanitizer formulations under controlled conditions.
• Facility Managers: For selecting disinfectant and sanitizer products that meet recognized standards for reducing microbial contamination on non-food contact surfaces.

Typical scenarios include hospitals, office buildings, schools, transportation, and other environments where maintaining hygienic non-food contact surfaces is critical for public health and safety.

Related Standards

• ASTM E2274: Method for evaluating laundry sanitizers and disinfectants.
• ASTM E1054: Practices for evaluation of inactivators (neutralizers) of antimicrobial agents.
• ASTM D1193: Specification for reagent water, required for preparation of dilution and testing solutions.
• ASTM E2756: Terminology relating to antimicrobial and antiviral agents.
• 40 CFR Part 160: Good Laboratory Practice (GLP) Standards, referenced for laboratory conduct and documentation procedures.

Practical Value

By adhering to ASTM E1153-22, organizations can:

• Ensure sanitizers meet internationally recognized benchmarks for effectiveness on hard, nonporous, non-food contact surfaces
• Provide credible, reproducible data supporting product claims for antimicrobial efficacy
• Meet regulatory requirements for product approval and market access
• Validate the performance of cleaner-sanitizer products designed for single-step cleaning and sanitizing of lightly soiled surfaces

This standard helps maintain consistent hygiene levels, reduce the risk of pathogen transmission, and uphold safety where surface hygiene is paramount. For additional technical detail or to ensure compliance, users should refer to the full official standard as published by ASTM International.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E1153 − 22
Standard Test Method for
Efficacy of Sanitizers Recommended for Inanimate, Hard,
Nonporous Non-Food Contact Surfaces
This standard is issued under the fixed designation E1153; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope* mendations issued by the World Trade Organization Technical
Barriers to Trade (TBT) Committee.
1.1 This test method is used to evaluate the antimicrobial
efficacy of sanitizers on precleaned, inanimate, hard,
2. Referenced Documents
nonporous, non-food contact surfaces against Staphylococcus
2.1 ASTM Standards:
aureus,or Klebsiella pneumoniaeor Klebsiella aerogenes,ora
D1193Specification for Reagent Water
combination thereof. Appropriate modifications to the method
E1054PracticesforEvaluationofInactivatorsofAntimicro-
may be required when testing organisms not specified herein.
bial Agents
When utilizing test surfaces not described herein (see Test
E2274Test Method for Evaluation of Laundry Sanitizers
Method E2274) or when evaluating spray-based or towelette-
and Disinfectants
based antimicrobial products, modifications may also be re-
E2756Terminology Relating toAntimicrobial andAntiviral
quired.
Agents
1.2 This test method may also be used to evaluate the
2.2 Federal Standard:
antimicrobial efficacy of one-step cleaner-sanitizer formula-
40CFR, Part 160, Good Laboratory Practice Standards
tions recommended for use on lightly soiled, inanimate,
nonporous, non-food contact surfaces. 3. Terminology
1.3 It is the responsibility of the investigator to determine 3.1 Terms used in this test method are defined in Terminol-
whether Good Laboratory Practices (GLP) are required and to ogy E2756.
follow them where appropriate (see section 40 CFR, 160 or as
3.2 Definitions of Terms Specific to This Standard:
revised.)
3.2.1 accuracy, n—ameasureofthedegreeofconformityof
1.4 The values stated in SI units are to be regarded as a value generated by a specific procedure to the assumed or
standard. No other units of measurement are included in this
accepted true value, and includes both precision and bias.
standard.
3.2.2 ambient temperature, n—temperature of the environ-
1.5 This standard may involve hazardous materials, chemi-
ment in which a test method is performed.
cals and microorganisms and should be performed only by
3.2.3 antimicrobial, adj—describes an agent that kills or
persons who have had formal microbiological training. This
inactivates microorganisms or suppresses their growth or
standard does not purport to address all of the safety concerns,
reproduction.
if any, associated with its use. It is the responsibility of the user
3.2.4 bias, n—a systematic error that contributes to the
of this standard to establish appropriate safety, health, and
difference between the mean of a large number of test results
environmental practices and determine the applicability of
and an accepted reference value.
regulatory limitations prior to use.
3.2.5 cleaner-sanitizer, n—a physical or chemical agent that
1.6 This international standard was developed in accor-
removes soil from an object and reduces numbers of microor-
dance with internationally recognized principles on standard-
ganisms on non-food contact surfaces.
ization established in the Decision on Principles for the
Development of International Standards, Guides and Recom- 3.2.6 carrier, n—a surrogate surface or matrix that facili-
tates the interaction of test microorganisms and treatment(s).
1 2
This test method is under the jurisdiction of ASTM Committee E35 on For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
responsibility of Subcommittee E35.15 on Antimicrobial Agents. Standards volume information, refer to the standard’s Document Summary page on
Current edition approved Oct. 1, 2022. Published October 2022. Originally the ASTM website.
ε1
approved 1987. Last previous edition approved in 2014 as E1153–03(2014) . Available from the Superintendent of Documents, U.S. Government Printing
DOI: 10.1520/E1153-22. Office, Washington, DC 20402.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1153 − 22
3.2.7 effıcacy, n—the proven performance of a product 5.11 Flaming Apparatus—A bunsen burner or other appro-
established under defined conditions of testing. priate heat sterilizer.
3.2.8 inoculum, n—the viable microorganisms used to con-
5.12 Mixer—A “vortex” mixer is recommended.
taminate a sample, device or surface, often expressed as to
5.13 Timer—A reliable stopwatch or laboratory timer ca-
number and type.
pable of measuring elapsed time in seconds and minutes.
3.2.9 neutralization, n—the process for inactivating or
5.14 pH Meter—Areliable, standardized pH meter to deter-
quenching the activity of a microbiocide, often achieved
mine pH of culture media.
through physical (for example, filtration or dilution) or chemi-
5.15 Desiccator, recommended size: 200 mm inside diam-
cal means.
eter with approximately 125mm chamber depth from inside
3.2.10 precision, n—the closeness of agreement between
plate to cover flange, glass.
independent test results obtained under prescribed conditions.
5.16 Incubator,capableofmaintainingtemperatureof25°C
3.2.11 reproducibility, n—the precision of test results ob-
to 32°C or 35°C to 39°C, or both.
tained in different laboratories performing the same test pro-
cedure under specifically defined conditions.
5.17 Sterilizer, steam sterilizer and hot air oven (≥180°C 6
2°C for ≥2 h).
3.2.12 sanitizer, n—chemical or physical agent(s) used to
reduce the number of microorganisms to a level judged to be
5.18 Colony Counter—Any one of several types may be
appropriate for a defined purpose and/or claim.
used, for example Quebec.
5.19 Membrane Filters, Compatible with the test organism
4. Significance and Use
(for example, 0.45 µm pore size).
4.1 Thistestmethodshallbeusedtodetermineifachemical
5.20 Filter Assembly, autoclavable or pre-sterilized.
intendedforuseasanon-foodcontactsanitizerorasaone-step
5.21 Forceps (may be autoclave sterilized prior to use).
cleaner-sanitizer provides percent reductions of the selected
test organisms on treated carriers as compared to control.
5.22 Refrigerator, capable of maintaining 2°C to 8°C.
5. Apparatus
6. Reagents and Materials
5.1 Balance—A calibrated balance with a platform to ac-
6.1 Purity of Reagents—Reagent grade chemicals shall be
commodate a 100mLvolumetric flask.This balance should be
used in all tests. Unless otherwise indicated, it is intended that
sensitive to 0.01 g.
all reagents shall conform to the specifications of the Commit-
5.2 Nonporous Test Surfaces, pre-cleaned.
tee onAnalytical Reagents of theAmerican Chemical Society,
5.2.1 Borosilicate Glass Squares, 25 by 25 by 2 mm slides,
where such specifications are available. Other grades may be
or 18 mm by 36 mm slides, nonchipped. 3 in. by 1 in. (76 mm
used, provided it is first ascertained that the reagent is of
by 25 mm) nonchipped slides may be used for towelette
sufficiently high purity to permit its use without lessening the
applications
accuracy of the determination.
5.2.2 Glazed Glass or Stainless Steel, of appropriate type,
6.2 Water for Dilution of Product Under Test:
approximately same size as in 5.2.1.
6.2.1 Water, sterile, deionized or distilled, equivalent to or
5.3 Glass Culture Tubes, recommended sizes: 18mm to
better than Type 3, see Specification D1193.
20mm by 150 mm and 25mm by 150 mm without lip.
6.2.2 Association of Offıcial Analytical Chemists (AOAC)
Synthetic Hard Water:
5.4 Culture Tube Closures, appropriate sized nontoxic clo-
6.2.2.1 Solution 1—Dissolve 31.74 g magnesium chloride
sures.
(MgCl ) (or equivalent of hydrates) and 73.99 g calcium
5.5 Pipets or Dispensing Syringes, (or both), appropriately
chloride (CaCl ) in boiled distilled or deionized water and
calibrated and sterile.
dilute to 1 L. Sterilize by autoclaving.
5.6 Bacteriological Transfer Loop, 4 mm inside diameter
6.2.2.2 Solution 2—Dissolve 56.03 g sodium bicarbonate
loop of platinum or platinum alloy wire or sterile, disposable
(NaHCO ) in boiled distilled or deionized water and dilute to
plastic loops of same size.
1 L. Sterilize by membrane filtration.
5.7 Flasks or Containers:
5.7.1 Appropriate sizes with closures for preparation of
culture medium and sterile deionized water. 4
Reagent Chemicals, American Chemical Society Specifications, American
5.7.2 Volumetric, 100mL and 1000 mL, sterile. Chemical Society, Washington, DC. For suggestions on the testing of reagents not
listed by the American Chemical Society, see Analar Standards for Laboratory
5.8 Petri dishes, recommended sizes: 50mm by 9 mm
Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
plastic, and 100 by 15 mm, glass and plastic; sterile. and National Formulary,U.S.PharmaceuticalConvention,Inc.(USPC),Rockville,
MD.
5.9 Jars, ointment jars, (for example polypropylene) 2 oz
“Official Methods of Analysis of the Association of Official Analytical
(60 mL), recommended, with nontoxic lids, sterile. Chemists,”AssociationofOfficialAnalyticalChemists,Washington,DC,Chapter6.
(a) Method 955.11 Section A. (a).
5.10 GraduatedCylinders,recommendedsizes;100mLand
(b) Method 955.11 Section A. (c).
500 mL. (c) Method 960.09 Section Sections D and E.
E1153 − 22
6.2.2.3 Place the desired amount of Solution 1 in a sterile 7.2.1 Test squares shall be dipped in acetone or 70% to
1L volumetric flask, or other appropriate volumetric vessel. 95% ethyl or isopropyl alcohol, rinsed with distilled or
Each1mLofSolution1willgiveawaterequivalenttoca.100 deionized water, and air dried before sterilization.
ppm of hardness calculated as calcium carbonate (CaCO)by 7.2.2 Place test squares into a large, glass dish and sterilize
theequationbelow.(Forexample,4mLofsolution1wouldbe in a hot air oven for ≥2hat ≥180°C.
added to the flask to target 400 ppm hardness in 1L of water.) 7.2.3 After sterilization, place each square into separate
Add approximately 600 mLor ⁄4 of the total water volume of 50mm by 9 mm or 100mm by 15 mm sterile plastic Petri
sterile distilled or deionized (reagent grade) water free of dishes using sterile technique.
substances that interfere with analytical methods; then add 4
8. Test Organisms
mLof Solution 2 and dilute to exactly 1 Lwith sterile distilled
or deionized water.
8.1 Klebsiella pneumoniae American Type Culture Collec-
tion (ATCC) 4352 or Klebsiella aerogenes American Type
TotalhardnessasppmCaCO (1)
Culture Collection (ATCC) 13048 and Staphylococcus aureus
5@2.495 3ppmCa#1@4.115 3ppmMg# ATCC 6538.
8.2 Maintenance of Test Organisms—Maintain stock cul-
6.2.3 The final pH of synthetic hard water should be from
tures on nutrient agar. Incubate 2 days at 35°C to 39°C for K.
7.6 to 8.0.
pneumoniae and S. aureus or 25°C to 32°C for K. aerogenes,
6.2.4 The synthetic water to be used for the testing should
then refrigerate at approximately 2°C to 8°C for up to one
be analyzed chemically for hardness at the time of test.
month(forexample,upto31days).Topreparethetestinocula,
Analysis may be performed by the method described in
transfer each culture for at least 3 days (transfers) as described
footnote5(c)orbycommerciallyavailablekit.Thewatermust
in 9.1. Stock slant cultures used for inoculation should not be
be used within 24 h of preparation but may be refrigerated at
more than five passages removed from the ATCC cultures
2°C to 8°C prior to use. The solution must be analyzed for
(USP XXIII). Information on long term culture maintenance
hardness on the day of use.
and storage is found in “Manual of Methods for General
6.2.5 Allwaterusedforpreparationoftestsolutionsshallbe
Bacteriology” and “ATCC Catalogue of Bacteria and Bacte-
sterile.
riophages”.
6.3 Sanitizing Solutions—Freshly prepared solutions of
sanitizers (for example, used within 8 h of dilution) shall be
9. Preparation of Inocula
used in all tests.
9.1 K. pneumoniae and S. aureus are grown in nutrient
6.4 Neutralizing Solutions—Solutions appropriate to inacti-
broth. K. aerogenes is grown in tryptic soy broth. From stock
vate sanitizing solutions shall be used in accordance with
cultures, (no more than 1 month old), inoculate tubes contain-
Practices E1054.
ing 10 mLof appropriate broth, and incubate for 24h 62hat
35°C to 39°C for K. pneumoniae and S. aureus or 25°C to
6.5 Culture Media:
(5(a))
32°C for K. aerogenes. Usinga4mm inside diameter transfer
6.5.1 Nutrient Broth.
(5(b))
loop, transfer a loopful of the culture into fresh broth. Make at
6.5.2 Nutrient Agar.
least three consecutive daily transfers prior to use as an
6.5.3 Tryptic Soy Broth, per manufacturer’s instructions
inoculum. The final transfer is incubated for 48 h to 54 h, and
6.5.4 Other appropriate growth medium or subculture agar
this culture is used for the test. Cultures may be appropriately
may be used where appropriate for the test organism (prepared
adjusted (by dilution with growth medium or centrifuge-
per manufacturer’s instructions or purchased commercially).
concentration) to ensure appropriate population control recov-
6.6 Soil, Fetal Bovine Serum, aseptically derived and main-
ery. Refer to 13.3.2 for the population control recovery
tained.
requirements.
9.2 Inocula for Testing Sanitizers for Use on Pre-cleaned
7. Preparation of Apparatus
Surfaces—Thoroughly mix 48h to 54 h culture of test organ-
7.1 Constant Humidity Chamber (Desiccator):
ism on “vortex” mixer, then allow the culture to settle for ≥15
7.1.1 At least one day prior to use, fill the lower portion of
min. Remove the upper two thirds of this suspension by
a large size desiccator with about 500 mLof glycerin solution
aspiration or decanting and use this as the inoculum for testing
having a refractive index of 1.4529 at 25°C (
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
´1
Designation: E1153 − 14 E1153 − 22
Standard Test Method for
Efficacy of Sanitizers Recommended for Inanimate, Hard,
Nonporous Non-Food Contact Surfaces
This standard is issued under the fixed designation E1153; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
ε NOTE—Section 13.3.2 was editorially corrected in June 2020.
1. Scope Scope*
1.1 This test method is used to evaluate the antimicrobial efficacy of sanitizers on precleaned, inanimate, hard, nonporous,
non-food contact surfaces against Staphylococcus aureus, or Klebsiella pneumoniae or EnterobacterKlebsiella aerogenes, or a
combination thereof. Appropriate modifications to the method may be required when testing organisms not specified herein. When
utilizing test surfaces not described herein (see Test Method E2274) or when evaluating spray-based or towelette-based
antimicrobial products, modifications may also be required.
1.2 This test method may also be used to evaluate the antimicrobial efficacy of one-step cleaner-sanitizer formulations
recommended for use on lightly soiled, inanimate, nonporous, non-food contact surfaces.
1.3 It is the responsibility of the investigator to determine whether Good Laboratory Practices (GLP) are required and to follow
them where appropriate (see section 40 CFR, 160 or as revised.)
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.5 This standard may involve hazardous materials, chemicals and microorganisms and should be performed only by persons
who have had formal microbiological training. This standard does not purport to address all of the safety concerns, if any,
associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental
practices and determine the applicability of regulatory limitations prior to use.
1.6 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
D1193 Specification for Reagent Water
E1054 Practices for Evaluation of Inactivators of Antimicrobial Agents
E2274 Test Method for Evaluation of Laundry Sanitizers and Disinfectants
This test method is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agents and is the direct responsibility of
Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved April 1, 2014Oct. 1, 2022. Published May 2014October 2022. Originally approved 1987. Last previous edition approved in 20102014 as
ε1
E1153 – 03(2010)(2014) . DOI: 10.1520/E1153-14E01.10.1520/E1153-22.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1153 − 22
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
2.2 Federal Standard:
40 CFR, Part 160, Good Laboratory Practice Standards
3. Terminology
3.1 Terms used in this test method are defined in Terminology E2756.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 accuracy, n—a measure of the degree of conformity of a value generated by a specific procedure to the assumed or accepted
true value, and includes both precision and bias.
3.2.2 ambient temperature, n—temperature of the environment in which a test method is performed.
3.2.3 antimicrobial, adj—describes an agent that kills or inactivates microorganisms or suppresses their growth or reproduction.
3.2.4 bias, n—a systematic error that contributes to the difference between the mean of a large number of test results and an
accepted reference value.
3.2.5 cleaner-sanitizer, n—a physical or chemical agent that removes soil from an object and reduces numbers of microorganisms
on non-food contact surfaces.
3.2.6 carrier, n—a surrogate surface or matrix that facilitates the interaction of test microorganisms and treatment(s).
3.2.7 effıcacy, n—the proven performance of a product established under defined conditions of testing.
3.2.8 inoculum, n—the viable microorganisms used to contaminate a sample, device or surface, often expressed as to number and
type.
3.2.9 neutralization, n—the process for inactivating or quenching the activity of a microbiocide, often achieved through physical
(for example, filtration or dilution) or chemical means.
3.2.10 precision, n—the closeness of agreement between independent test results obtained under prescribed conditions.
3.2.11 reproducibility, n—the precision of test results obtained in different laboratories performing the same test procedure under
specifically defined conditions.
3.2.12 sanitizer, n—chemical or physical agent(s) used to reduce the number of microorganisms to a level judged to be appropriate
for a defined purpose and/or claim.
4. Significance and Use
4.1 This test method shall be used to determine if a chemical intended for use as a non-food contact sanitizer or as a one-step
cleaner-sanitizer provides percent reductions of the selected test organisms on treated carriers as compared to control.
5. Apparatus
5.1 Balance—A calibrated balance with a platform to accommodate a 100-mL100 mL volumetric flask. This balance should be
sensitive to 0.01 g.
5.2 Nonporous Test Surfaces, pre-cleaned.
Available from the Superintendent of Documents, U.S. Government Printing Office, Washington, DC 20402.
E1153 − 22
5.2.1 Borosilicate Glass Squares, 25 by 25 by 2 mm slides, or 18 mm by 36 mm slides, nonchipped. 3 in. by 1 in. (76 mm by
25 mm) nonchipped slides may be used for towelette applications
5.2.2 Glazed Glass or Stainless Steel, of appropriate type, approximately same size as in 5.2.1.
5.3 Glass Culture Tubes, recommended sizes: 1818 mm to 2020 mm by 150 mm and 2525 mm by 150 mm without lip.
5.4 Culture Tube Closures, appropriate sized nontoxic closures.
5.5 Pipets or Dispensing Syringes, (or both), appropriately calibrated and sterile.
5.6 Bacteriological Transfer Loop, 4 mm inside diameter loop of platinum or platinum alloy wire or sterile, disposable plastic
loops of same size.
5.7 Flasks or Containers:
5.7.1 Appropriate sizes with closures for preparation of culture medium and sterile deionized water.
5.7.2 Volumetric, 100100 mL and 1000 mL, sterile.
5.8 Petri dishes, recommended sizes: 5050 mm by 9 mm plastic, and 100 by 15 mm, glass and plastic; sterile.
5.9 Jars, ointment jars, (for example polypropylene) 2 oz (60 mL), recommended, with nontoxic lids, sterile.
5.10 Graduated Cylinders, recommended sizes; 100100 mL and 500 mL.
5.11 Flaming Apparatus—A bunsen burner or other appropriate heat sterilizer.
5.12 Mixer—A “vortex” mixer is recommended.
5.13 Timer—A reliable stopwatch or laboratory timer capable of measuring elapsed time in seconds and minutes.
5.14 pH Meter—A reliable, standardized pH meter to determine pH of culture media.
5.15 Desiccator, recommended size: 200 mm inside diameter with approximately 125-mm125 mm chamber depth from inside
plate to cover flange, glass.
5.16 Incubator, capable of maintaining temperature of 2525 °C to 32°C32 °C or 3535 °C to 39°C,39 °C, or both.
5.17 Sterilizer, steam sterilizer and hot air oven (≥180(≥180 °C 6 2°C2 °C for ≥2 h).
5.18 Colony Counter—Any one of several types may be used, for example Quebec.
5.19 Membrane Filters, Compatible with the test organism (for example, 0.45 μm pore size).
5.20 Filter Assembly, autoclavable or pre-sterilized.
5.21 Forceps (may be autoclave sterilized prior to use).
5.22 Refrigerator, capable of maintaining 22 °C to 8°C.8 °C.
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6. Reagents and Materials
6.1 Purity of Reagents—Reagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that all
reagents shall conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society, where
such specifications are available. Other grades may be used, provided it is first ascertained that the reagent is of sufficiently high
purity to permit its use without lessening the accuracy of the determination.
6.2 Water for Dilution of Product Under Test:
6.2.1 Water, sterile, deionized or distilled, equivalent to or better than Type 3, see Specification D1193.
5(c)
6.2.2 Association of Offıcial Analytical Chemists (AOAC) Synthetic Hard Water:
6.2.2.1 Solution 1—Dissolve 31.74 g magnesium chloride (MgCl ) (or equivalent of hydrates) and 73.99 g calcium chloride
(CaCl ) in boiled distilled or deionized water and dilute to 1 L. Sterilize by autoclaving.
6.2.2.2 Solution 2—Dissolve 56.03 g sodium bicarbonate (NaHCO ) in boiled distilled or deionized water and dilute to 1 L.
Sterilize by membrane filtration.
6.2.2.3 Place the desired amount of Solution 1 in a sterile 1-L1 L volumetric flask, or other appropriate volumetric vessel. Each
1 mL of Solution 1 will give a water equivalent to ca. 100 ppm of hardness calculated as calcium carbonate (CaCO ) by the
equation below. (For example, 4 mL of solution 1 would be added to the flask to target 400 ppm hardness in 1L of water.) Add
approximately 600 mL or ⁄4 of the total water volume of sterile distilled or deionized (reagent grade) water free of substances that
interfere with analytical methods; then add 4 mL of Solution 2 and dilute to exactly 1 L with sterile distilled or deionized water.
Total hardness as ppm CaCO (1)
5@2.495 3ppm Ca#1@4.115 3ppm Mg#
6.2.3 The final pH of synthetic hard water should be from 7.6 to 8.0.
6.2.4 The synthetic water to be used for the testing should be analyzed chemically for hardness at the time of test. Analysis may
be performed by the method described in footnote 5(c) or by commercially available kit. The water must be used within 24 h of
preparation but may be refrigerated at 22 °C to 8°C8 °C prior to use. The solution must be analyzed for hardness on the day of
use.
6.2.5 All water used for preparation of test solutions shall be sterile.
6.3 Sanitizing Solutions—Freshly prepared solutions of sanitizers (for example, used within 8 h of dilution) shall be used in all
tests.
6.4 Neutralizing Solutions—Solutions appropriate to inactivate sanitizing solutions shall be used in accordance with Practices
E1054.
6.5 Culture Media:
(5(a))
6.5.1 Nutrient Broth.
Reagent Chemicals, American Chemical Society Specifications, American Chemical Society, Washington, DC. For suggestions on the testing of reagents not listed by
the American Chemical Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia and National
Formulary, U.S. Pharmaceutical Convention, Inc. (USPC), Rockville, MD.
“Official Methods of Analysis of the Association of Official Analytical Chemists,” Association of Official Analytical Chemists, Washington, DC, Chapter 6.
(a) Method 955.11 Section A. (a).
(b) Method 955.11 Section A. (c).
(c) Method 960.09 Section Sections D and E.
(a) Method 955.11 Section A. (a).
(b) Method 955.11 Section A. (c).
(c) Method 960.09 Section Sections D and E.
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(5(b))
6.5.2 Nutrient Agar.
6.5.3 Tryptic Soy Broth, per manufacturer’s instructions
6.5.4 Other appropriate growth medium or subculture agar may be used where appropriate for the test organism (prepared per
manufacturer’s instructions or purchased commercially).
6.6 Soil, Fetal Bovine Serum, aseptically derived and maintained.
7. Preparation of Apparatus
7.1 Constant Humidity Chamber (Desiccator):
7.1.1 At least one day prior to use, fill the lower portion of a large size desiccator with about 500 mL of glycerin solution having
a refractive index of 1.4529 at 25°C25 °C (approximately 86.5 % glycerin in distilled water will provide this refractive index). This
will provide a constant 4040 % to 41 % relative humidity at 3535 °C to 39°C39 °C in which the inoculated nonporous square
surfaces will be dried prior to treatment with the sanitizer. Replace the porcelain floor plate of the desiccator and store at 3535 °C
to 39°C39 °C to allow to come to equilibrium. Alternatively, a humidity controlled incubator set to 35 to 39°C may be used to
achieve drying conditions appropriate for maximum survival of the test organism.
7.2 Test Squares:
7.2.1 Test squares shall be dipped in acetone or 7070 % to 95 % ethyl or isopropyl alcohol, rinsed with distilled or deionized water,
and air dried before sterilization.
7.2.2 Place test squares into a large, glass dish and sterilize in a hot air oven for ≥2 h at ≥180°C.≥180 °C.
7.2.3 After sterilization, place each square into separate 5050 mm by 9 mm or 100100 mm by 15 mm sterile plastic Petri dishes
using sterile technique.
8. Test Organisms
8.1 Klebsiella pneumoniae American Type Culture Collection (ATCC) 4352 or EnterobacterKlebsiella aerogenes American Type
Culture Collection (ATCC) 13048 and Staphylococcus aureus ATCC 6538.
8.2 Maintenance of Test Organisms—Maintain stock cultures on nutrient agar. Incubate 2 days at 3535 °C to 39°C39 °C for K.
pneumoniae and S. aureus or 2525 °C to 32°C32 °C for E.K. aerogenes, then refrigerate at approximately 22 °C to 8°C8 °C for
up to one month (for example, up to 31 days). To prepare the test inocula, transfer each culture for at least 3 days (transfers) as
described in 9.1. Stock slant cultures used for inoculation should not be more than five passages removed from the ATCC cultures
(USP XXIII). Information on long term culture maintenance and storage is found in “Manual of Methods for General
7 8
Bacteriology” and “ATCC Catalogue of Bacteria and Bacteriophages”.
9. Preparation of Inocula
9.1 K. pneumoniae and S. aureus are grown in nutrient broth. E.K. aerogenes is grown in tryptic soy broth. From stock cultures,
(no more than 1 month old), inoculate tubes containing 10 mL of appropriate broth, and incubate for 2424 h 6 2h2 h at 3535 °C
to 39°C39 °C for K. pneumoniae and S. aureus or 2525 °C to 32°C32 °C for E.K. aerogenes. Using a 4 mm inside diameter transfer
loop, transfer a loopful of the culture into fresh broth. Make at least three c
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